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The transfer of human artificial chromosomes via cryopreserved microcells
Narumi Uno1, Katsuhiro Uno, Susi Zatti
1Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan.
Abstract:
Microcell-mediated chromosome transfer (MMCT) technology enables a single and intact mammalian chromosome or megabase-sized chromosome fragments to be transferred from donor to recipient cells. The conventional MMCT method is performed immediately after the purification of microcells. The timing of the isolation of microcells and the preparation of recipient cells is very important. Thus, ready-made microcells can improve and simplify the process of MMCT. Here, we established a cryopreservation method to store microcells at -80 °C, and compared these cells with conventionally- (immediately-) prepared cells with respect to the efficiency of MMCT and the stability of a human artificial chromosome (HAC) transferred to human HT1080 cells. The HAC transfer in microcell hybrids was confirmed by FISH analysis. There was no significant difference between the two methods regarding chromosome transfer efficiency and the retention rate of HAC. Thus, cryopreservation of ready-to-use microcells provides an improved and simplified protocol for MMCT.
Insights
Cryopreservation of microcells offers a simplified approach to microcell-mediated chromosome transfer (MMCT). Stored microcells demonstrate comparable efficiency and stability for human artificial chromosome (HAC) transfer to recipient cells.
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- Microcell-mediated chromosome transfer (MMCT) is a key technology for transferring mammalian chromosomes.
- Current MMCT protocols require immediate use of microcells after isolation, demanding precise timing.
- Streamlining MMCT requires methods for preparing and storing microcells in advance.
Purpose of the Study:
- To establish and evaluate a cryopreservation method for microcells.
- To compare the efficiency and stability of MMCT using cryopreserved versus freshly prepared microcells.
- To assess the retention of human artificial chromosomes (HACs) in recipient cells.
Main Methods:
- Development of a cryopreservation protocol for microcells stored at -80°C.
- Comparison of MMCT efficiency using cryopreserved and immediately prepared microcells.
- FISH analysis to confirm HAC transfer and assess retention in HT1080 cells.
Main Results:
- Cryopreservation of microcells at -80°C is feasible.
- No significant difference in MMCT efficiency was observed between cryopreserved and fresh microcells.
- Comparable retention rates of human artificial chromosomes (HACs) were found in recipient cells.
Conclusions:
- Cryopreservation of microcells provides a viable and simplified alternative for MMCT.
- Ready-to-use cryopreserved microcells enhance the practicality of chromosome transfer protocols.
- This method facilitates more flexible and accessible applications of MMCT technology.
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